WO2009058168A1 - Fibronectine cellulaire en tant que marqueur de diagnostic dans une maladie cardiovasculaire et ses procédés d'utilisation - Google Patents

Fibronectine cellulaire en tant que marqueur de diagnostic dans une maladie cardiovasculaire et ses procédés d'utilisation Download PDF

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WO2009058168A1
WO2009058168A1 PCT/US2008/008967 US2008008967W WO2009058168A1 WO 2009058168 A1 WO2009058168 A1 WO 2009058168A1 US 2008008967 W US2008008967 W US 2008008967W WO 2009058168 A1 WO2009058168 A1 WO 2009058168A1
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algorithms
risk
bleeding
markers
protein
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Cornelius Diamond
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Prediction Sciences Llc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • the present invention generally relates to the identification and use of diagnostic markers for vascular damage leading to bleeding events in cardiovascular disease, particularily myocardial infarction (Ml).
  • the present invention particularly relates to methods for (1) the prediction of a bleeding event in cardiac patients prior to surgery; (2) the prediction of intracerebral hemorrhage (ICH) in cardiac patients who are given a thrombolytic; (3) for the idenification of patients who would not have an elevated bleeding risk when given a combination antiplatlet/ thienopyridine derivative therapy such as aspirin or dipyridamole, and clopidogrel or ticlopidine, and (4) the identification of patients who could benefit from cardiac artery stenting or balloon angioplasty.
  • ICH intracerebral hemorrhage
  • thrombolytic therapy with fibrinolytic (thrombolytic) agents has revolutionized the treatment of diverse circulatory disorders such as pulmonary embolism, deep-vein thrombosis and myocardial infarction [see for instance Collen D, Stump DC, Gold HK (1988) Thrombolytic therapy. Annu Rev Med 39:405-423].
  • These circulatory disorders are increasingly becoming the leading causes of mortality in modern societies worldwide.
  • Thrombolytic agents have the unique ability to activate the components intrinsic to the fibrinolytic system, resulting in the degradation of blood clots, which restores blood flow through the occluded vessels [see for instance Collen D, Lijnen JR (1986) The fibrinolytic system in man. Crit Rev Hemat Oncol 4:249-301].
  • the fibrinolytic agents commonly used in thrombolytic therapy are streptokinase (SK), urokinase (UK) and derivatives of tissue type plasminogen activator (tPA or Alteplase) such as Reteplase, Tenecteplase, and Lanoteplase.
  • SK streptokinase
  • UK urokinase
  • tPA tissue type plasminogen activator
  • PG enzymatically inert plasminogen
  • PN active protease
  • thrombolytic agent during therapy is dictated by a number of factors, which depends essentially upon the relative merits and demerits of individual PG activators. These include the cost of the drug, the side-effects and their severity such as major bleeding and lntercerebral hemorrhage (ICH), in vivo stability and specificity towards fibrin clots and immunological reactivity.
  • ICH major bleeding and lntercerebral hemorrhage
  • balloon angioplasty reduces short- term death, nonfatal myocardial infarction, and stroke when compared with thrombolytic reperfusion [see for instance E. C. Keeley, J.A. Boura and CL. Grines, Primary angioplasty versus intravenous thrombolytic therapy for acute myocardial infarction: a quantitative review of 23 randomised trials. Lancet 361 (2003), pp. 13-20.]. Still, the clinical efficacy of balloon angioplasty is limited by the development of late restenosis in up to 50% of patients, and by recurrent myocardial infarction in 3% to 5% of patients [see for instance CM. Nunn, W.W.
  • Reteplase (recombinant plasminogen activator, r-PA) is a single chain deletion mutant of alteplase that is expressed in Escherichia coli and, therefore, is expressed as an unglycosylated protein.
  • Reteplase includes 355 amino acids with a total molecular weight of 39 kDa. The molecule consists of cringle 2 and the protease domain of thereteplase molecule. Because of the deletion of the fibronectin finger region, the binding of reteplase to fibrin is significantly reduced in comparison with that of alteplase.
  • reteplase is stimulated in the presence of fibrin to a lower extent than alteplase, suggesting that the fibronectin finger is involved in the stimulation of the protease as well.
  • Reteplase in comparison with alteplase, is characterised by reduced fibrin selectivity.
  • reteplase and alteplase do not differ with respect to their activity as plasminogen activators, nor do they differ with respect to their inhibition by the plasminogen activator inhibitor type 1 (PAI-1). (see for instance Martin U, Bader R, B ⁇ hm E, et al.
  • BM 06.022 a novel recombinant plasminogen activator. Cardiovasc Drugs Rev 1993;11 :299-311.).
  • reteplase in comparison with alteplase is equal in efficacy and superior in its application as a double bolus that also facilitates prehospital initiation of reperfusion therapy.
  • Tenecteplase is also called the TNK-mutant of alteplase.
  • the molecule does not constitute a deletion mutant of alteplase (as reteplase does). Instead, it consists of the alteplase molecule with the exception of three point mutations.
  • At position 103 of the polypeptide the aminoacid threonine has been replaced by asparagine leading to a new glycosylation site.
  • the carbohydrate chain that is linked to this site enlarges the molecule, thereby reducing its elimination and prolonging its plasma half life.
  • Asparagine has been replaced by glutamine. By the exchange of this amino acid the carbohydrate side chain that facilitates hepatic elimination has been removed. Hence, plasma half life is further prolonged.
  • Tenecteplase has been tested extensively in clinical trials.
  • ASSENT-1 assessment of safety and efficacy of a new thrombolytic agent
  • ASSENT-1 trial in patients with acute myocardial infarction(see for instance van de Werf F et al.
  • Am Heart J 1999;137:786-91. single bolus tenecteplase proved to be as safe as the gold standard of thrombolytic therapy, the accelerated regimen of alteplase (initial bolus followed by an infusion over 90 minutes).
  • the plasminogen activator lanoteplase (novel plasminogen activator, n- PA) is another deletions mutant of thereteplase molecule that also exhibits an additional, single point mutation.
  • the fibronectin finger region and the epidermal growth factor domain have been deleted in the lanoteplase molecule.
  • the aminoacid asparagine has been replaced by glutamine. Because of this point mutation the glycosylation site that is responsible for facilitated hepatic elimination is lost. Consequently, the plasma half life of lanoteplase is increased.
  • lanoteplase is expressed in CHO cells, just as is tenecteplase.
  • CHO cells are eukaryotic cells, in contrast to prokaryotic cells such as E coli.
  • the plasma half life of lanoteplase is about 10 times that of alteplase and may reach 45 minutes.
  • lanoteplase can be administered as a single bolus. This molecule is described further in U.S. patent 06/882,051 filed July 3, 1986, incorporated by reference.
  • the Gp llb/llla inhibitors form the most potent antiplatelet therapy now available. Among them abciximab, eptifibatide, and tirofiban have proven their clinical efficacy.
  • TIMI 14 trial alteplase at half dose (15 mg as an initial bolus, 35 mg as an infusion over 60 minutes) combined with abciximab at full dose (0.25 mg/kg as an initial bolus, 10 ⁇ g/min as an infusion over 12 hours) yielded the highest patency rate 90 minutes after the initiation of treatment (TIMI 3 flow in 76% of treated patients) without increasing the risk of severe bleeding complications (see Antman EM et al. Abciximab facilitates the rate and extent of thrombolysis. Results of the TIMI 14 trial. Circulation 1999;99:2710-32.).
  • Major bleeds defined as retroperitoneal, intracerebral, or fatal bleedings with the need for vascular repair or blood transfusion, are also a major problem in the administration of thrombolytic therapy.
  • major bleeds occurred at a rate of 5.9% for accelerated infusion of alteplase and 4.65% for tenecteplase in cardiac patients given a thrombolytic.
  • the present invention relates to the identification and use of diagnostic markers for the detection of bleed events in cardiovascular disease.
  • the methods and compositions described herein can meet a need in the healing arts for rapid, sensitive and specific diagnostic assay to be used in the diagnosis and differentiation of various bleeding events.
  • the methods and compositions of the present invention can also be used to facilitate the treatment of Ml patients and the development of additional diagnostic and/or prognostic indicators.
  • the present invention relates to (1) materials and procedures for identifying markers that are associated with the diagnosis, prognosis, or differentiation of patients at risk of a major bleeding event or ICH following a cardiovascular event such as Ml; (2) using such markers in diagnosing and treating a patient with cardiovascular disease and/or monitoring the course of a treatment regimen; (3) using such markers to identify subjects at risk for one or more adverse outcomes related to Ml and/or may benefit from a therapy that would not normally be given to patients due to extra-ordinary risk of a major bleed event or ICH by providing a prediction as to bleed or ICH risk for the indivual patient, said patient who is predicted to have a greatly lessened risk of a major bleed event or ICH when given said therapy is able to use said therapy; and (4) using at one of such markers an outcome marker for screening compounds and pharmaceutical compositions that might provide a benefit in treating or preventing such conditions.
  • the invention discloses methods for determining a prediction of a bleeding event such as ICH in patients suffering from cardiovascular disease.
  • the preferred method includes analyzing a fluid sample obtained from a person who has an unknown risk for the levels of one or more markers specific to the damage caused by said cardiovascular disease.
  • these markers would be drawn from the group consisting of markers relating to vascular damage, glial activation, inflammatory mediation, thrombosis, cellular injury, apoptosis, myelin breakdown, and specific and non-specific markers of cardiovascular disease.
  • the analysis of the preferred method thus more precisely includes identifying one or more markers the presence or amount of which is associated with the diagnosis, prognosis, or differentiation of cardiovascular events, prediction of major bleeding events or ICH, and/or efficacy of a therapeutic treatment for cardiovascular disease.
  • markers the level of such marker(s) in a sample obtained from a subject of interest can be measured.
  • these markers can be compared to a level that is associated with the diagnosis, prognosis, or differentiation of cardiovascular disease including prediction of bleeding or ICH risk or suitability of admistration of a therapeutic such as lanoteplase to a patient.
  • the instant invention is embodied in methods for choosing one or more marker(s) for prediction of bleeding and/or ICH risk in patients with cardiovascular disease, including determination of ICH risk before admistering a specific therapy such as lanoteplase, that together, and as a group, have maximal sensitivity, specificity, and predictive power.
  • Said maximal sensitivity, specificity, and predictive power is in particular realized by choosing one or more markers as constitute a group by process of plotting receiver operator characteristic (ROC) curves for (1) the sensitivity of a particular combination of markers versus (2) specificity for said combination at various cutoff threshold levels.
  • ROC receiver operator characteristic
  • the instant invention further discloses methods to interpolate the nonlinear correlative effects of one or more markers chosen by any methodology to such that the interaction between markers of said combination of one or more markers promotes maximal sensitivity, specificity, and predictive accuracy in the prediction of any of the occurrence of major bleed events, ICH, determination of thrombolytic usage, and/or prediction of bleeding risk in stenting and/or angioplasty.
  • markers refers to proteins or polypeptides to be used as targets for screening test samples obtained from subjects.
  • Proteins or polypeptides used as markers in the present invention are contemplated to include any fragments thereof, in particular, immunologically detectable fragments.
  • proteins which are released by cells of the central nervous system which become damaged during a cerebral attack could become degraded or cleaved into such fragments.
  • certain markers are synthesized in an inactive form, which may be subsequently activated, e.g., by proteolysis. Examples of such markers are described hereinafter.
  • related marker refers to one or more fragments of a particular marker that may be detected as a surrogate for the marker itself.
  • These related markers may be, for example, “pre,” “pro,” or “prepro” forms of markers, or the "pre,” “pro,” or “prepro” fragment removed to form the mature marker.
  • Exemplary markers that are synthesized as pre, pro, and prepro forms are described hereinafter. In preferred embodiments, these "pre,” “pro,” or “prepro” forms or the removed “pre,” “pro,” or “prepro” fragments are used in an equivalent fashion to the mature markers in the methods described herein.
  • Preferred markers of the invention can aid in the determination of risk of a major bleeding event or ICH, and/or predicting subsequent enlargement of the hematoma after ICH.
  • Preferred markers are drawn from the group including c-Fn, MMP-9, myelin basic protein, IL-1 , IL-1 r ⁇ , IL-1 ⁇ , IL-6, IL-8, IL-10, NCAM, VCAM, ICAM, S100 ⁇ , GFAP, BNGF, CRP, ⁇ -TG, PF-4, D-Dimer, TGF- ⁇ , NT-3, F 1+2 , VEGF, CK-BB, caspase 3, MCP-1 , total homocysteine (tHcy), thrombin- antithrombin III complex, tissue factor, GFAP, NSE- ⁇ , vWF, VEGF, FPA, serum amyloid A (SAA), and NR2A/2B.
  • markers from this group are ones that have proven highly predictive of hemorrhagic events in the area of ischemic stroke: namely, cellular fibronectin (c-Fn) and matrix metalloprotein-9 (MMP-9).
  • marker levels vary at certain time points; for example, the level of a marker may be at one level at three hours post-cardiovascular event, and another level at nine hours post- cardiovascular event.
  • This algorithm in current state of the art is a simple threshold level above which a marker is said to be indicative of an adverse event in the human body.
  • a particular diagnosis and/or prognosis of said adverse event may depend upon the comparison of each marker to this value; alternatively, if only a subset of markers are outside of a normal range, then this subset may be indicative of a said adverse event.
  • a plurality of markers are combined using an algorithm to increase the predictive value of the analysis in comparison to that obtained from the markers taken individually or in smaller groups.
  • one or more markers for vascular damage, glial activation, inflammatory mediation, thrombosis, cellular injury, apoptosis, myelin breakdown, and specific and non-specific markers of cardiovascular disease are combined in a single assay to enhance the predictive value of the described methods.
  • This assay is usefully predictive of multiple outcomes, for instance: determining whether or not a hemorrhagic event occurred, determining the suitability of a certain, then further predicting stroke prognosis.
  • different marker combinations in the assay may be used for different indications.
  • different algorithms interpret the marker levels as indicated on the same assay for different indications.
  • Preferred panels comprise markers for the following purposes in patients suffering from cardiovascular disease: (1) prediction of ICH risk; (2) prediction of major bleed events; (3) prediction of bleeding risk from thrombolytic use, particularily lanoteplase; (4) diagnosis of cardiovascular disease and indication if a hemorrhagic or bleeding event has occurred; and (5) determination of whether stenting or angioplasty is preferrable in light of risk of a bleeding complication.
  • particular thresholds for one or more markers in a panel are not relied upon to determine if a profile of marker levels obtained from a subject are indicative of a particular diagnosis/prognosis. Rather, in accordance with the present invention, an evaluation of the entire profile is made by (1) first training an algorithm with marker information from samples from a test population and a disease population to which the clinical outcome of interest has occurred to determine weighting factors for each marker, and (2) then evaluating that result on a previously unseen population. Certain persons skilled in bioinformatics will recognize this procedure to be tanatamount to the construction, and to the training, of a machine learning algorithm such as a kemal partial least squares algorithm.
  • the evaluation is determined by maximizing the numerical area under the ROC curve for the sensitivity of a particular panel of markers versus specificity for said panel at various individual marker levels. From this number, the skilled artisan can then predict a probability ⁇ that a subject's current marker levels in said combination is indicative of the clinical marker of interest.
  • the test population might consist solely of samples from a group of subjects who have had cardiovascular disease and no other comorbid disease conditions, while (2) the disease population might consist solely of samples from a group of subjects who have had a hemorrhagic event after treatment of a Ml that said group of subjects had all experienced.
  • a third, "normal" population might also be used to establish baseline levels of markers as well in a non-diseased population.
  • the aforementioned weighting factors are multiplicative of marker levels in a nonlinear fashion.
  • Each weighting factor is a function of other marker levels in the panel combination, and consists of terms that relate individual contributions, or independent and correlative, or dependent, terms. In the case of a marker having no interaction with other markers in regards to then clinical outcome of interest, then the specific value of the dependent terms would be zero.
  • the present invention is embodied in methods for determining a treatment regimen for use in a patient who has experienced a cardiovascular event, particularily Ml.
  • the methods preferably comprise determining a level of one or more diagnostic or predictive markers as described herein, and using the markers to determine a treatment course for a patient.
  • a prediction might include the likelyhood of IHC after Ml when administering a thrombolytic therapy such as lanoteplase.
  • One or more treatment regimens that improve the patient's prognosis by reducing the increased disposition for an adverse outcome associated with the prognosis can then be used to treat the patient.
  • Such methods preferably comprise comparing an amount of a marker predictive of a subsequent bleed event or ICH after Ml, said marker selected from the group consisting of cellular fibronectin (c-Fn), and matrix metalloprotease-9 (MMP-9), in a test sample from a patient diagnosed with acute Ml to a predictive level of said marker, wherein said patient is identified as being at risk for a subsequent bleed event or ICH after Ml by a level of said marker equal to or greater than said predictive level.
  • the present invention is embodied in kits for determining the diagnosis or prognosis of a patient. These kits preferably comprise devices, software and reagents for measuring one or more marker levels in a patient sample, and instructions for performing the assay.
  • kits contain a computer software program to be run on a computer or other means for converting marker level(s) to a prognosis.
  • kits preferably contain sufficient reagents to perform one or more such determinations, and are standardized to run on an instrument used to analyze blood samples, such as Abbott Laboratories' AxSYM®, Roche Diagnostics' Cardiac Reader®, or Dade Behring's Stratus® CS Analyzer.
  • the kits may be packaged with a corresponding therapeutic, such as lanoteplase, with the administration of the drug cleared if the test kit delivers a result that is below a pre-determined threshold that predicts a particular adverse event, such as ICH, when the patient is administered said therapeutic.
  • test sample refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, or evaluation of a subject of interest, such as a patient. In certain embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition.
  • Preferred test samples include blood, serum, plasma, cerebrospinal fluid, urine and saliva.
  • one of skill in the art would realize that some test samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.
  • markers of glial activation refers to markers that indicate glial cell function. Glia mediate neuroendocrine and neuroimmune functions and are also important in synaptic remodeling and the loss of synaptic connections that occur during aging. These functions are carried out by changes in glia, including changes in shape, interactions with neurons and other glia, and gene expression. The predominant change that occurs in glia during aging is glial activation, which can progress to reactive gliosis in response to neurodegeneration. Markers distinguish normal and reactive glia. During aging, astrocytes hypertrophy and exhibit signs of metabolic activation, and astrocytic processes surround neurons.
  • Microglia also become activated and subsets of activated microglial increase in number and may enter the phagocytic or reactive stage. Yet glial cells are intimately involved in the biochemical metabolic and neurotrophic support of the function of neurons, and glial actions at the synapses are crucial to normal neuronal transmission. Glia take up excess glutamate (which can be neurotoxic) and produce neurotrophic factors which keep cells alive, as well as interacting with other systems in transmitter-like actions. Thus, a loss of normal glial function could have dramatic impacts on normal neuronal function. Such specific markers of glial activation include, but are not limited to, GFAP, S100B, Mac-1 , TLR4, TGF- ⁇ 1 and CD14.
  • markers of vascular damage refers to markers that indicate endothelial damage.
  • endothelium When the endothelium is damaged or becomes dysfunctional, a cascade leading to atherogenesis is precipitated, initiating a cycle of injury, immunologic induction, and amplification.
  • Dysfunctional endothelium leads to increased permeability to lipoproteins and up- regulation of leukocyte and endothelial adhesion molecules.
  • certain activating substances including oxidized LDL, monocyte chemotactic protein 1 , interleukin (IL)-8, and platelet-derived growth factor (PDGF), leukocytes migrate into the wall of the artery.
  • Such specific markers of vascular damage include, but are not limited to, c-Fn, MMP-9, endothelin-1 (ET- 1), von Willebrand factor (vWf), and soluble (S-) adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), plasma indexes of endothelial damage/dysfunction and soluble thrombomodulin (sTM).
  • markers of inflammatory mediation refers to markers that indicate an inflammatory response to a cardiovascular event. Inflammatory responses are initiated and perpetuated by the interaction of immune cells with cells of the affected vessel wall. This is directed by a network of chemical messengers, which, in a state of vascular health, exist as balanced but opposing forces. These markers include various cytokines, proteases, adhesion molecules, and acute phase proteins as participants in the generation of vascular inflammation.
  • Such specific markers of vascular damage include, but are not limited to, Cellular adhesion molecules such as Intracellular adhesion molecule-1 , Vascular cellular adhesion molecule- 1 , NCAM and Selectins such as E-Selectin; Chemokines such as monocyte chemoattractant protein-1 ; Cytokines such as lnterleukins 1 , 1 ⁇ , 1 receptor antagonist, 6, 8, 10, 18, transforming growth factor ⁇ , and Tumor necrosis factor- ⁇ ; Proteases such as the matrix metalloproteinases MMP-9, MMP-3, and MMP- 2; Accessory signaling markers such as CD40/CD40L; and acute phase proteins such as C-reactive protein, vascular endothelial growth factor, ceruloplasmin, fibrinogen, ⁇ 1-acid glycoprotein, ⁇ 1 -antitrypsin, and haptoglobin.
  • Cellular adhesion molecules such as Intracellular adhesion molecule-1 , Vascular cellular adhe
  • markers of thrombosis refers to markers that indicate an coagulation event in ischaemic stroke.
  • the blood clotting system is activated when blood vessels are damaged, exposing collagen, the major protein that connective tissue is made from. Platelets circulating in the blood adhere to exposed collagen on the cell wall of the blood vessel and secrete chemicals that start the clotting process as follows: Platelet aggregators cause platelets to clump together (aggregate). They also cause the blood vessels to contract (vasoconstrict), which reduces blood loss. Platelet aggregators include adenosine diphosphate (ADP), thromboxane A2, and serotonin (5-HT).
  • ADP adenosine diphosphate
  • thromboxane A2 thromboxane A2
  • serotonin 5-HT
  • Fibrin is formed from fibrinogen in a complex series of reactions called the coagulation cascade.
  • the enzymes that comprise the coagulation system are called coagulation factors, which are numbered in the order in which they were discovered. They include factor XII 1 factor Xl, factor IX, factor X, factor VII, and factor V.
  • the activation of the coagulation factors results in the formation of thrombin, which acts as a cofactor for the conversion of fibrinogen into fibrin.
  • Platelet aggregation inhibitors and vasodilators such as nitric oxide and prostacyclin, which is also known as prostaglandin 12 (PGI2)
  • PKI2 prostaglandin 12
  • Plasminogen activators that promote the breakdown of fibrin such as tissue plasminogen activator (t-PA)
  • t-PA tissue plasminogen activator
  • Anticoagulants that inhibit enzymes in the coagulation cascade such as antithrombin III (activated by heparin) and proteins C and S.
  • Such specific markers of thrombosis include, but are not limited to, von Willebrand factor, thrombin-antithrombin III complex, proteins C and S, tissue factor, fibrinopeptide A, plasmin- ⁇ -2-antiplasmin complex, prothrombin fragment 1+2, D-dimer, platelet factor 4, and ⁇ -thromboglobulin.
  • the term "marker of cellular injury and myelin breakdown” as used in this specification refers to markers associated with damage to the structural and functional molecules of the cell. Although any biologically important molecule in a cell can be the target of injury producing stress, four biochemical systems are particularly vulnerable: (1) the cell membrane, (2) energy metabolism, (3) protein synthesis, and (4) genes. Because many of the biochemical systems of the cell are inter-dependent, injury at one site typically leads to secondary injury to other cellular processes.
  • Myelin is the outer lipid rich (fatty) layer that covers nerves and nervous system pathways in the brain and spinal cord.
  • the myelin sheath a lipid-rich multilamellar membrane of relative stability, both insulates and enhances conduction in nerve axons.
  • a notable feature of myelin-specific proteins, in particular myelin basic protein, is their susceptibility to proteolytic activity and their encephalitogenicity, which induces inflammatory demyelination in the CNS.
  • the final common pathway of myelin breakdown in vivo is well documented and there is evidence that myelin disruption can be mediated directly by soluble (circulating) factors and for following receptor-driven phagocytosis by macrophages.
  • the exact mechanism(s) of demyelination in ischemic attack is still unresolved, both antigen-specific and-non-specific events having the potential to generate the myelinolytic process.
  • Cerebral injury leads to breakdown of the blood-brain barrier (BBB), exposing CNS antigens to the peripheral circulation and allowing the peripheral circulation access to the brain.
  • BBB blood-brain barrier
  • the breakdown of the BBB leads to rapid acquisition of MBP-reactive T cell clones and lgs in stroke patients, but does not lead to autoimmune encephalitis.
  • MBP myelin basic protein
  • the degradation of myelin basic protein (MBP) by proteinase yields encephalitogenic peptides and its loss has been found to cause structural alteration of the myelin sheath. This suggests that MBP degradation is an initial step in the breakdown of myelin in demyelinating diseases.
  • the source of increased proteinase activity has been indicated as macrophages, lymphocytes, and proliferative astrocytes (reactive cells).
  • Increased proteinase activity is found in Schwann cells in Wallehan degeneration, and the presence of calpain in myelin-forming oligodendrocytes and Schwann cells suggests that these cells are likely sources of degradative enzymes.
  • Such specific markers of cellular injury and myelin breakdown include, but are not limited to, creatinine phosphokinase brain band, tissue factor, Proteolipid protein, RU Malendialdehyde, calpain, and myelin basic protein.
  • the term "marker of apoptosis or growth factors" as used in this specification refers to markers involved in neuronal cell death. Numerous studies in experimental models of ischemia have now reported that apoptosis contributes to neuronal death (reviewed by Chalmers-Redman et al Mechanisms of nerve cell death: apoptosis or necrosis after cerebral ischemia. In: Green AR, Cross AJ, eds. Neuroprotective Agents and Cerebral Ischemia . San Diego, Calif: Academic Press; 1997:1-25.). Apoptosis requires the activation of a "cell death" gene program, and many of the extracellular signals that regulate apoptosis have been identified.
  • Fas/APO-1 a cell surface protein, with its ligand (Fas-L) leads to programmed cell death.
  • Soluble (s) Fas/APO-1 a molecule lacking the transmembrane domain of Fas/APO-1 , blocks apoptosis by inhibiting interaction between Fas/APO-1 and Fas-L on the cell surface (see for instance Cheng J et al., Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science. 1994;263:1759- 1762.). Fas expression has been detected on B and T cells and on neutrophils.
  • Fas/Fas-L pathway is one of the major mechanisms for T-cell-mediated cytotoxicity. It has also been demonstrated by in situ hybridization that the expression of Fas/APO-1 was induced in murine brain after transient global cerebral ischemia. Another gene product, bcl-2, has been shown to suppress apoptosis and to protect primary neuronal cell cultures from apoptosis induced by nerve growth factor depletion.
  • Macrophages and T lymphocytes kill target cells by inducing apoptosis, one of the potential mechanisms whereby the inflammatory cells invading the infarcted brain area participate in neuronal cell death.
  • Stroke patients displayed an intrathecal production of proinflammatory cytokines, such as interleukin (IL)- 1 ⁇ , IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF), and of the anti-inflammatory cytokine IL-10 within the first 24 hours after the onset of symptoms, supporting the notion of localized immune response to the acute brain lesion in humans.
  • proinflammatory cytokines such as interleukin (IL)- 1 ⁇ , IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF)
  • TNF- ⁇ a powerful cytokine inducing apoptosis in the extraneural compartment of the body, has been demonstrated to protect rat hippocampal, septal, and cortical cells against metabolic-excitotoxic insults and to facilitate regeneration of injured axons. More importantly, TNF- ⁇ and - ⁇ protect neurons against amyloid ⁇ -protein-triggered toxicity.
  • caspases a unique family of structurally related, highly conserved, aspartate- specific, cysteine proteases that are necessary to carry out the signal for apoptotic cell death.
  • caspase-1 and caspase-3 Two members of the caspase family, caspase-1 and caspase-3, are known to cleave the most abundant caspase target substrate, actin.
  • actin The 45-kDa actin is cleaved by caspase activation between Asp11 and Asn12 and between Asp244 and Gly245 to produce N-terminal 32-kDa fragments and C-terminal 15-kDa fragments.
  • a polyclonal antibody to the last 5 amino acids of the C-terminus of the 32-kDa fragment of actin generated by caspase cleavage of intact actin has been developed and named "fractin” for "fragment of actin.”
  • Fractin labeling provides indirect evidence of caspase activation and demonstrates initiation of an apoptotic pathway, but does not rule out secondary necrosis.
  • Other markers for apoptosis include biochemical evidence of oligointemucleosomal DNA fragmentation into approximately 180-bp multiples resulting from endonuclease activation that can be demonstrated with a typical "laddering" appearance on agarose gel electrophoresis.
  • TUNEL terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling
  • Such specific markers of apoptosis and growth factors include, but are not limited to, Brain natriuretic peptide, caspase 3, calbindin-D, heat shock protein 60 and 70, c-fos, c-jun, ubiquitin, and cytochrome C.
  • specific marker of cardiovascular disease refers to proteins or polypeptides that are associated with cardiovascular and heart tissue, and which can be correlated with a cardiovascular disease, but are not correlated with other types of disease.
  • specific markers of cardiovascular disease include, but are not limited to, troponin I, troponin T, Heart-fatty acid-binding protein, CRP, D-Dimer, tHcy, SAA, microalbuminuria, aldosterone, PAI-1 , myeloperoxidase, proteolipid protein, thrombomodulin, and lipoprotein-associated phospholipase A2 (Lp-PLA2).
  • non-specific marker of cardiovascular disease refers to proteins or polypeptides that are elevated in the event of cardiovascular disease, but may also be elevated due to non-cardiovascular events.
  • Non-specific markers include, but are not limited to, ApoC-l and ApoC-ll, A-type natriuretic peptide, B-type natriuretic peptide, C-type natriuretic peptide, adrenomedullin, ⁇ - thromboglobulin, C-reactive protein, Cardiac Troponin I and Troponin T, Creatine kinase MB, D-dimer, E-selectin, endothelin- 1 , endothelin-2, and endothelin-3, A- , F- , and H- Fatty acid binding protein, fibhnopeptide A, hemoglobin ⁇ 2l chain head activator, insulin-like growth factor-1 , MMP-3,
  • diagnosis refers to predict the type of disease or condition from a set of marker values and/or patient symptoms. This is in contrast to disease prediction, which is to predict the occurrence of disease before it occurs, and the term “prognosis”, which is to predict disease progression at a future point in time from one or more indicator value(s) at a previous point in time.
  • prognosis which is to predict disease progression at a future point in time from one or more indicator value(s) at a previous point in time.
  • correlating refers to a process in which a set of examples of clinical inputs from subjects, such as marker levels, and their corresponding outputs, such as whether a subject suffered from a specific type of stroke, are related to each other.
  • This relationship can be determined by comparing such examples to examples from a control and/or disease-free population at a later point in time, and selecting those indicators which can differentiate between the two disease states as a function of time alone or in combination at a certain probability level.
  • the selection process is described herein.
  • the selected markers each at a certain level range which might be a simple threshold, are said to be correlative or associative with one of the disease states. Said correlated markers can be then be used for disease detection, diagnosis, prognosis and/or treatment outcome.
  • Preferred methods of correlating markers is by performing marker selection by a feature selection algorithm and classification by mapping functions described herein.
  • a preferred probability level is a 3% chance, 5% chance, a 7% chance, a 10% chance, a 15% chance, a 20% chance, a 25% chance, a 30% chance, a 35% chance, a 40% chance, a 45% chance, a 50% chance, a 55% chance, a 60% chance, a 65% chance, a 70% chance, a 75% chance, a 80% chance, a 85% chance, a 90% chance, a 95% chance, and a 100% chance.
  • Each of these values of probability is plus or minus 2% or less.
  • a preferred threshold level for markers of the present invention is about 25 pg/mL, about 50 pg/mL, about 60 pg/mL, about 75 pg/mL, about 100 pg/mL, about 150 pg/mL, about 200 pg/mL, about 300 pg/mL, about 400 pg/mL, about 500 pg/mL, about 600 pg/mL, about 750 pg/mL, about 1000 pg/mL, about 2500 pg/mL, about 0.15 ⁇ g/mL, about 2 ⁇ g/mL, about 3.5 ⁇ g/mL, about 5.5 ⁇ g/mL, and about 6 ⁇ g/mL.
  • the term "about” in this context refers to +/-10%.
  • Fibronectins are adhesive dimeric glycoproteins that promote cell-cell and cell-matrix interactions (see for instance Hynes RO. Fibronectins. Sci Am. 1986;254:42-51.).
  • Plasma fibronectin (p-Fn) is primarily produced by hepatocytes, but plasma also contains small quantities of cellular fibronectin (c- Fn), which is mainly synthesized by endothelial cells (see for instance Peters JH 1 Sporn LA, Ginsberg MH, Wagner DD. Human endothelial cells synthesize, process, and secrete fibronectin molecules bearing an alternatively spliced type Il homology (ED1). Blood. 1990;75: 1801 -1808.).
  • c-Fn is largely confined to the vascular endothelium, high plasma levels of this molecule might be indicative of endothelial damage.
  • plasma c-Fn levels have been reported to be increased in patients with vascular injury secondary to vasculitis, sepsis, acute major trauma, and diabetes, (see for instance Peters JH, Maunder RJ, Woolf AD, Cochrane GH, Ginsberg MH. Elevated plasma levels of ED1_ ("cellular”) fibronectin in patients with vascular injury. J Lab CHn Med. 1989; 113:586-597; Kanters SD, Banga JD, Algra A, Frijns RC, Beutler JJ, Fijnheer R.
  • Cellular Fibronectin or ED1+. is an adhesive glycoprotein, is a fibronectin synthesized in endothelial cells. It contains an extra Type III domain (ED1 , or EDA/EIIIA), as a result of alternative mRNA splicing. It circulates in the blood in small quantities. Endothelial cells do not express the ED1 domain under normal circumstances, but the ED1 domain is included in fibronectin molecules in pathological conditions (see for instance Dubin D, Peters JH, Brown LF, Logan B, Kent KC, Berse B, Berven S, Cercek B, Sharifi BG, Pratt RE: Balloon catheterization induced arterial expression of embryonic fibronectins.
  • ICH has a 30-50% mortality rate, half of this coming from continued bleeding, identification of markers of such bleeds is also of critical importance to change treatment outcomes.
  • the instant invention demonstrates that plasma c- Fn levels in patients experiencing a Ml are significantly higher in patients in which ICH occurs following thrombolytic therapy and teaches that c-Fn levels >6 ⁇ g/ml_ can predict the development of IHC with a sensitivity and negative predictive value of 100%. Therefore, c-Fn is a useful marker of those patients who are at greatest risk for ICH after Ml.
  • Intracerebral hemorrhage triggers interleukin-6 and interleukin-10 release in blood. Stroke. 2002;33:2334-2335.; Beamer NB, Coull BM, Clark WM, Hazel JS, Silberger JR. Interleukin-6 and interleukin-1 receptor antagonist in acute stroke. Ann Neurol. 1995; 37:800-805.; Montaner J, Alvarez-Sabin J, Molina C, et al. Matrix metalloproteinase expression after human cardioembolic stroke: temporal profile and relation to neurological impairment. Stroke. 2001 ;32:1759-1766.; Perini F, Morra M, Alecci M, Galloni E, Marchi M, Toso V.
  • No. 11/338,447 relates to the use of levels of an NMDA receptor peptide or antibody such as NR2a for aiding in the assessment of the risk of stroke in an apparently healthy human subject prior to surgery. Again, this invention is concerning a different molecule than the ones described in the instant invention. Additionally, the type of stroke as is apparent in the claims of U.S. patent application Ser. No. 11/338,447 is ischemic, not hemorrhagic stroke, and the usage of the molecular test is before surgery such as surgery after Ml, not the administration of a therapeutic, such as the administration of a thrombolytic after Ml.
  • the instant invention provides a methodology to predict bleeding and ICH risk in cardiovascular patients that are treated therapeutically or with a device to restore antegrade flow in the infarct vessel or prevent atherosclerotic buildup.
  • Clinical information such as sex, age, time from onset of symptoms to treatment, NIHSS score, biochemistry and vital signs at admission, and neuroimaging findings are collected at various time periods.
  • Preferred time periods for the instant invention include 0, 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 24 hours, 36 hours, 48 hours, 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 3 months and 6 months.
  • Time is measured either from onset of symptoms or admission into a clinical setting where the patient receives care.
  • This marker and clinical information form a set of examples of clinical inputs and their corresponding outputs, the outputs being the clinical outcome of interest, for instance stroke and stroke subtype occurrence or non-occurrence, or occurrence and type of major bleed.
  • the therapeutic used to restore antegrade flow in the infarct vessel or prevent atherosclerotic buildup is also noted. These quantities are as described in the Introduction.
  • the classifer is trained, it is ready for use by a clinician.
  • the clinician enters the same classifer inputs used during training of the network by assaying the selected markers and collecting relevant clinical information for a new patient, and the trained classifier outputs a maximum likelihood estimator for the value of the output given the inputs for the current patient.
  • the clinician or patient can then act on this value.
  • immunoassay devices and methods are often used. These devices and methods can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of an analyte of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule.
  • the markers are analyzed using an immunoassay, although other methods are well known to those skilled in the art (for example, the measurement of marker RNA levels).
  • the presence or amount of a marker is generally determined using antibodies specific for each marker and detecting specific binding. Any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassay (RIAs), competitive binding assays, and the like. Specific immunological binding of the antibody to the marker can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.
  • Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase and the like.
  • RAMP Biomedical device called the Clinical Reader sup.TM.
  • the fluoresent tag method used as the RAMP Biomedical device. Diluted whole blood is applied to the sample well. The red blood cells are retained in the sample pad, and the separated plasma migrates along the strip. Fluorescent dyed latex particles bind to the analyte and are immobilized at the detection zone. Additional particles are immobilized at the internal control zone.
  • the fluorescence of the detection and internal control zones are measured on the RAMP Clinical Reader sup.TM., and the ratio between these values is calculated. This ratio is used to determine the analyte concentration by interpolation from a lot-specific standard curve supplied by the manufacturer in each test kit for each assay.
  • immobilized antibodies specific for the markers is also contemplated by the present invention and is well known by one of ordinary skill in the art.
  • the antibodies could be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay place (such as microtiter wells), pieces of a solid substrate material (such as plastic, nylon, paper), and the like.
  • An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
  • the analysis of a plurality of markers may be carried out separately or simultaneously with one test sample. Several markers may be combined into one test for efficient processing of a multiple of samples. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same individual. Such testing of serial samples will allow the identification of changes in marker levels over time. Increases or decreases in marker levels, as well as the absence of change in marker levels, would provide useful information about the disease status that includes, but is not limited to identifying the approximate time from onset of the event, the presence and amount of salvagable tissue, the appropriateness of drug therapies, the effectiveness of various therapies, identification of the severity of the event, identification of the disease severity, and identification of the patient's outcome, including risk of future events.
  • An assay consisting of a combination of the markers referenced in the instant invention may be constructed to provide relevant information related to differential diagnosis.
  • a panel may be constucted using 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more or individual markers, though a number lower than 4 markers is the most preferred embodiment.
  • the analysis of a single marker or subsets of markers comprising a larger panel of markers could be carried out methods described within the instant invention to optimize clinical sensitivity or specificity in various clinical settings.
  • the clinical sensitivity of an assay is defined as the percentage of those with the disease that the assay correctly predicts, and the specificity of an assay is defined as the percentage of those without the disease that the assay correctly predicts (Tietz Textbook of Clinical Chemistry, 2.sup.nd edition, Carl Burtis and Edward Ashwood eds., W. B. Saunders and Company, p. 496).
  • markers could be carried out in a variety of physical formats as well.
  • the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples.
  • single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings.
  • Particularly useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different analytes.
  • Such formats include protein microarrays, or "protein chips" (see, e.g., Ng and Hag, J. Cell MoI. Med. 6: 329-340 (2002)) and capillary devices.
  • the present invention provides a kit for the analysis of markers.
  • a kit preferably comprises devises and reagents for the analysis of at least one test sample and instructions for performing the assay.
  • the kits may contain one or more means for using information obtained from immunoassays performed for a marker panel to rule in or out certain diagnoses.
  • Marker antibodies or antigens may be incorporated into immunoassay diagnostic kits depending upon which marker autoantibodies or antigens are being measured.
  • a first container may include a composition comprising an antigen or antibody preparation. Both antibody and antigen preparations should preferably be provided in a suitable titrated form, with antigen concentrations and/or antibody titers given for easy reference in quantitative applications.
  • kits may also include an immunodetection reagent or label for the detection of specific immunoreaction between the provided antigen and/or antibody, as the case may be, and the diagnostic sample.
  • Suitable detection reagents are well known in the art as exemplified by radioactive, enzymatic or otherwise chromogenic ligands, which are typically employed in association with the antigen and/or antibody, or in association with a second antibody having specificity for first antibody.
  • the reaction is detected or quantified by means of detecting or quantifying the label.
  • Immunodetection reagents and processes suitable for application in connection with the novel methods of the present invention are generally well known in the art.
  • the reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like.
  • the diagnostic kit may further include where necessary agents for reducing background interference in a test, agents for increasing signal, software and algorithms for combining and interpolating marker values to produce a prediction of clinical outcome of interest, apparatus for conducting a test, calibration curves and charts, standardization curves and charts, and the like.
  • kits for detecting various markers indicative of bleed or ICH risk prediction comprising: (1) an immunosorbent for selected markers indicative of bleed or ICH risk prediction, and (2) an indicator reagent comprising secondary antibodies attached to a signal generating compound for each individual marker.
  • the secondary antibodies can be specific for each individual marker or for the primary antibodies in the immunosorbent.
  • the kits further comprise an immunosorbent for glutamate or polyglutamate, and/or an immunosorbent for homocysteine or polyhomocysteine, and secondary antibodies against the glutamate and/or homocysteine, or to the primary antibodies on the immunosorbents against the glutamate or homocysteine.
  • the immunosorbent preferably comprises anti-antibodies for the biomarkers bound to a solid support.
  • the liquid is then removed by aspiration, decanting or any other convenient method the particles are washed and dispersed in a volume of a suitable buffer that is smaller than the volume of the original sample.
  • the target molecule on their outer surface binds to its binding partner in the immovable stripe, causing superparamagnetic particles to accumulate along the stripe.
  • immovable striping of binding partner for the target molecule multiple lines, spaced apart from one another along the end of the strip remote from the sample receiving end, may be appropriate to ensure efficient capture of the target ligand in this assay.
  • the magnetic signal of the superparamagnetic tag on the capture line or lines in millivolts, is read in a suitable instrument.
  • the present invention relates to a test-kit that relies upon PCR amplification for measuring selected markers indicative of bleed or ICH risk prediction.
  • the invention provides a kit comprising: (a) one or more oligonucleotide primers attached to a solid phase, (b) indicator reagent attached to a signal-generating compound capable of generating a detectable signal from oligonucleotides, and (c) a control sample (i.e. template cDNA).
  • the reagents may also include ancillary agents such as buffering agents, polymerase agents, and the like.
  • test-kit comprises (a) a solid phase to which biological fluids for receiving total DNA including selected marker cDNA indicative of bleed or ICH risk prediction could be attached, (b) oligonucleotide primers, preferably in a ready-to-use PCR buffer, and (c) a control sample (i.e. template cDNA).
  • a control sample i.e. template cDNA
  • the invention provides a diagnostic kit for detecting selected markers indicative of bleed or ICH risk prediction autoantibodies comprising (a) a polypeptide of the selected markers indicative of bleed or ICH risk prediction, fragment thereof, or analog or derivative thereof, (b) an indicator reagent comprising a secondary antibody specific for the autoantibody or the polypeptide attached to a signal-generating compound; and (c) a control sample, such as a known concentration of said selected markers indicative of bleed or ICH risk prediction diagnosis polyclonal antibodies.
  • the reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like.
  • the diagnostic kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme and nonenzyme substrates), agents for reducing background interference in a test, agents to increase the signal, apparatus for conducting a test, calibration and standardization information or instructions, and the like.
  • other members of the signal-producing system of which system the detectable group is a member e.g., enzyme and nonenzyme substrates
  • agents for reducing background interference in a test agents to increase the signal
  • apparatus for conducting a test e.g., calibration and standardization information or instructions, and the like.
  • Non-linear techniques for data analysis and information extraction are important for identifying complex interactions between markers that contribute to overall presentation of the clinical outcome.
  • association studies such as the one proposed
  • the construction of these in-silico predictors is a complex process. Often one must consider more markers to test than samples, missing values, poor generalization of results, selection of free parameters in predictor models, confidence in finding a sub- optimal solution and others.
  • the process for building a predictor is as important as designing the protocol for the association studies. Errors at each step can propagate downstream, affecting the generalizability of the final result.
  • data may be obtained from a group of subjects.
  • the subjects may be patients who have been tested for the presence or level of certain markers.
  • markers and methods of patient extraction are well known to those skilled in the art.
  • a particular set of markers may be relevant to a particular condition or disease. The method is not dependent on the actual markers.
  • the markers discussed in this document are included only for illustration and are not intended to limit the scope of the invention. Examples of such markers and panels of markers are described in the instant invention and the incorporated references.
  • a preferred embodiment of the instant invention is that the samples come from two or more different sets of patients, one a disease group of interest and the other(s) a control group, which may be healthy or diseased in a different indication than the disease group of interest. For instance, one might want to look at the difference in blood-borne markers between patients who have had an ICH following thrombolytic treatment for Ml and those who did not have any bleeding or ICH following thrombolytic treatment for Ml to differentiate between the two populations.
  • the blood samples are assayed, and the resulting set of values are put into a database, along with outcome, also called phenotype, information detailing the side effect of treatment, for instance ICH, once this is known. Additional clinical details such as time from onset of symptoms and patient physiological, medical, and demographics, the sum total called patient characteristics, are put into the database.
  • the time from onset is important to know as initial marker values from onset of symptoms can change significantly over time on a timeframe of tens of minutes. Thus, a marker may be significant at one point in the patient history and not at another in predicting diagnosis or prognosis of cardiovascular disease, damage or injury.
  • the database can be simple as a spreadsheet, i.e. a two-dimensional table of values, with rows being patients and columns being filled with patient marker and other characteristic values.
  • a computerized algorithm can first perform preprocessing of the data values. This involves normalization of the values across the dataset and/or transformation into a different representation for further processing. The dataset is then analyzed for missing values. Missing values are either replaced using an imputation algorithm, in a preferred embodiment using KNN or MVC algorithms, or the patient attached to the missing value is exised from the database. If greater than 50% of the other patients have the same missing value then value can be ignored. [0095] Once all missing values have been accounted for, the dataset is split up into three parts: a training set comprising 33-80% of the patients and their associated values, a testing set comprising 10-50% of the patients and their associated values, and a validation set comprising 1-50% of the patients and their associated values.
  • a feature selection algorithm is applied to the training dataset. This feature selection algorithm selects the most relevant marker values and/or patient characteristics. Preferred feature selection algorithms include, but are not limited to, Forward or Backward Floating, SVMs, Markov Blankets, Tree Based Methods with node discarding, Genetic Algorithms, Regression-based methods, kernel-based methods, and filter-based methods.
  • Cross-validation is one of several approaches to estimating how well the features selected from some training data is going to perform on future as-yet-unseen data and is well-known to the skilled artisan.
  • Cross validation is a model evaluation method that is better than residuals. The problem with residual evaluations is that they do not give an indication of how well the learner will do when it is asked to make new predictions for data it has not already seen.
  • One way to overcome this problem is to not use the entire data set when training a learner. Some of the data is removed before training begins. Then when training is done, the data that was removed can be used to test the performance of the learned model on " new" data.
  • the algorithm can optimize these selected markers by applying a classifer to the training dataset to predict clinical outcome.
  • a cost function that the classifier optimizes is specified according to outcome desired, for instance an area under receiver-operator curve maximizing the product of sensitivity and specificity of the selected markers, or positive or negative predictive accuracy.
  • Testing of the classifier is done on the testing dataset in a cross-validated fashion, preferably naive or k-fold cross- validation. Further detail is given in U.S. patent application 09/611 ,220, incorporated by reference.
  • Classifiers map input variables, in this case patient marker values, to outcomes of interest, for instance, prediction of stroke subtype.
  • Preferred classifiers include, but are not limited to, neural networks, Decision Trees, genetic algorithms, SVMs, Regression Trees, Cascade Correlation, Group Method Data Handling (GMDH), Multivariate Adaptive Regression Splines (MARS), Multilinear Interpolation, Radial Basis Functions, Robust Regression, Cascade Correlation + Projection Pursuit, linear regression, Non-linear regression, Polynomial Regression, Regression Trees, Multilinear Interpolation, MARS, Bayes classifiers and networks, and Markov Models, and Kernel Methods.
  • neural networks Decision Trees, genetic algorithms, SVMs, Regression Trees, Cascade Correlation, Group Method Data Handling (GMDH), Multivariate Adaptive Regression Splines (MARS), Multilinear Interpolation, Radial Basis Functions, Robust Regression, Cascade Correlation + Projection Pursuit, linear regression, Non-linear regression, Polynomial Regression, Regression Trees, Multilinear Interpolation
  • the classification model is then optimized by for instance combining the model with other models in an ensemble fashion.
  • Preferred methods for classifier optimization include, but are not limited to, boosting, bagging, entropy-based, and voting networks.
  • This classifier is now known as the final predictive model.
  • the predictive model is tested on the validation data set, not used in either feature selection or classification, to obtain an estimate of performance in a similar population.
  • the predictive model can be translated into a decision tree format for subdividing the patient population and making the decision output of the model easy to understand for the clinician.
  • the marker input values might include a time since symptom onset value and/or a threshold value. Using these marker inputs, the predictive model delivers diagnositic or prognostic output value along with associated error.
  • the instant invention anticipates a kit comprised of reagents, devices and instructions for performing the assays, and a computer software program comprised of the predictive model that interprets the assay values when entered into the predictive model run on a computer.
  • the predictive model receives the marker values via the computer that it resides upon.
  • FIGURE 1 is a table illustrating clinical parameters among a set of patients who suffered an Ml and a stroke after administration of thrombolytic therapy and a set of patients who did not suffer a stroke when given thrombolytic therapy;
  • FIGURE 2 is a Kruskill-Wallis plot showing c-Fn plasma molecular levels between cardioembolic stroke patients/patients who suffered an ischemic stroke after an Ml event and who did and did not suffer an ICH after administration of thrombolytic therapy and a set of non-cardioembolic stroke patients who did and did not suffer an ICH when given thrombolytic therapy;
  • FIGURE 3 is a scatter plot showing MMP-9 values divided by 30 on the x- axis plotted against c-Fn values on the y-axis for cardiac patients who suffered a Ml and a bleeding event after administration of thrombolytic therapy.
  • -9 is no data available
  • -1 is unknown outcome
  • 0 normals healthy controls
  • 1 is HI-1 events
  • 2 is HI-2 events
  • 3 is PH-1 events
  • 4 is PH-2 events.
  • ICH was diagnosed when the National Institutes of Health Stroke Scale score worsened by >4 points between baseline and 24 hours, and confirmed by CT scan. CT scans were performed immediately before treatment and at 24 to 36 hours after thrombolytic therapy or on neurologic deterioration.
  • HT was classified as hemorrhagic infarction type 1 or type 2, and PH, as type 1 , type 2, or remote PH.
  • relevant HT was defined as hemorrhagic infarction type 2 and any type of PH.
  • HT was defined as symptomatic when it was associated with early neurologic deterioration.
  • Lesion volumes were calculated on the radiographic plate using the formula 0.5 x a x b x c (where a is the maximal longitudinal diameter, b is the maximal transverse diameter, and c is the number of 10-mm slices containing hemorrhage).
  • the volume of the ICH plus that of the zone of peripheral hypodensity was determined using the same volumetric method described; the absolute volume of the hypodensity was calculated by subtracting the volume of the ICH from that of the total lesion (ICH plus peripheral hypodensity).

Abstract

Une thérapie thrombolytique dans le traitement d'un événement cardiovasculaire tel qu'un infarctus du myocarde (MI) contient un risque de souffrir d'un incident hémorragique conduisant à un handicap grave et souvent à la mort. Des procédés pour l'évaluation d'une thérapie correcte pour un patient spécifique qui a souffert d'un événement cardiovasculaire emploient une diversité de biomarqueurs comprenant la fibronectine cellulaire (c-Fn) assemblés en tant que panel pour l'évaluation. L'invention porte sur des procédés pour sélectionner des marqueurs et corréler leurs niveaux combinés avec un résultat clinique d'intérêt. Sous divers aspects, les procédés permettent une détection précoce d'événements de saignement potentiel, la détermination du pronostic d'un patient présentant un dommage cardiovasculaire, et l'identification d'un patient à risque d'une hémorragie lorsqu'on lui donne une thérapie thrombolytique. Les procédés décrits fournissent des dosages rapides, sensibles et spécifiques pour réduire grandement le risque de saignement ou le nombre de patients qui peuvent recevoir le traitement le plus utile pour leur événement cardiovasculaire et pour réduire les coûts humains et économiques associés à un saignement à la suite de tels traitements.
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WO2013188828A1 (fr) 2012-06-15 2013-12-19 Harry Stylli Méthodes de détection de maladies ou d'états au moyen de cellules infectées en circulation
WO2013188846A1 (fr) 2012-06-15 2013-12-19 Harry Stylli Procédés de détection de maladies ou d'états
JP2015514986A (ja) * 2012-04-13 2015-05-21 プレディクション ビオシアンス ソシエテ パ アクシオンス シンプリフィエ 細胞フィブロネクチンについての迅速な検査
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